The goal of the proposed research is to determine the site and mechanism of action of botulinum toxin. This goal will be pursued by focusing on three areas of research closely related to current knowledge on the structure and function of the toxin molecule. Botulinum toxin acts on cholinergic nerve endings to block transmitter release. To exert this action, the toxin proceeds through a sequence of three steps: 1.) a membrane binding step, 2.) an internalization step, and 3.) an intracellular poisoning step. The precise mechanism of intracellular poisoning remains unknown, but the extraordinary potency of the molecule suggests that it is an enzyme. The proposed model for botulinum toxin action was developed on murine preparations (phrenic nerve hemidiaphragm), and it has been partially confirmed on other laboratory animal preparations (e.g., PC-12 cells, adrenal chromaffin cells, pituitary cells). However, botulinum toxin action has never been studied on isolated human neuromuscular junctions. Therefore, the first task of the proposed research will be to determine the sequence of events involved in poisoning of human tissues. Evidence from laboratory animal studies indicates that botulinum toxin acts inside cells to block exocytosis. It is anticipated that the same result will be obtained with human cells. However, there are no data that identify the specific domain within the toxin molecule that is responsible for intracellular poisoning. Therefore, the second task of the proposed research will be to identify a specific polypeptide that blocks exocytosis. There is a prevailing belief that botulinum toxin is an enzyme, and thus that it catalytically modifies a substrate essential for transmitter release. However, neither the location of the substrate nor its role in exocytosis has been determined. Therefore, the final task of the proposed research will be to identify an intracellular target molecule that interacts with, or is enzymatically modified by, botulinum toxin.